Ras and Ras-related, small molecular weight GTPases function in the regulation of signaling and cell growth control and collectively serve to control cell proliferation, differentiation and apoptosis. There are over 150 known human proteins belonging to the Ras superfamily of GTPases. When mutant or hyperactivated, Ras family members contribute to oncogenesis and hereditary disorders affecting vision, immune and neurologic function. Strategies for inhibiting Ras to date have relied on altering membrane recruitment with drugs affecting prenylation. Inhibition of prenylation enzymes lacks specificity and is problematic because the cellular prenylation machinery is required for the proper function of many Ras superfamily members. The demonstrated efficacy of targeting drugs to the nucleotide binding pocket of specific kinases offers a paradigm that may be applied to the GTPases. The PI and co-I have established a fluorescent GTP-binding assay for GST-GTPase chimeras that can be monitored by flow cytometry. In addition, through outreach, the new Mexico Molecular Libraries Screening Center has established the ability to screen multiple GTPases simultaneously with multiplex analysis on a HyperCyt??high throughput flow cytometer. Advantages of the multiplex approach include single step analysis of drug specificity and selectivity, small reaction volumes and the discrimination of free and bound fluorescence that is conservative with respect to reagent usage when compared to polarization assays. The group has in hand a collection of small Ras-related GTPases as GST fusion proteins that include members of the Rab, Rho, and Ras families. It is the aim of the present application to identify lead compounds that interfere with fluorescent GTP-binding to individual Ras superfamily members. Subsequent functional assays will be used to identify the compounds as direct and allosteric inhibitors or activators of GTPase function. The identification of small molecule nucleotide binding modulators of small GTPases has not yet been undertaken and is expected to have important utility in the future treatment of cancer and neurologic diseases where GTPase function is specifically altered. [unreadable] [unreadable] [unreadable]